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Research on the cloning and analyzing of the human collagen gene

人类胶原蛋白基因的克隆与分析研究

基本信息

批准号：
03670178
负责人：
YOSHIOKA Hidekatsu
金额：
$ 1.34万
依托单位：
Okayama Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1993
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03670178/
关键词：
Collagen Extracellular Matrix cDNA Gene コラ-ゲン

项目摘要

1) Analysis of the promoter of the human alpha1(XI) collagen geneWe have cloned the 5' portion of the human COL11A1 gene and determined the location of its major start site of transcription. Analysis of the immediate upstream sequence revealed the lack of a TATA motif and the presence of several GC boxes. Consistent with this finding, primer extension and RNase protection assays identified several minor transcriptional start sites around a major one, 318-bp upstream from the ATG codon. Transient transfection experiments has showed the 1.6kb upstream region has the ability of the promoter. Deletion constructs narrowed down the sequence of its transcriptional activity between nucleotides -541 to -199. In this region, there is a single foot printing area containing a sequence with similarity to an AP-3 binding site.2) Primary structure and gene expression of a new human collagen chainTo determine the complete primary structure of the chain, we have isolated overlapping clones from a random-primed cDNA library from human rhabdomyosarcoma cell line (CCL 136) mRNA.The deduced polypeptide contained an open reading frame of 1142 amino acid residues. The predicted polypeptide consists of five collagenous domains of the COL1 to COL5. Structurally, this collagen chain most resembles to the alpha1(XVI) chain. Northern blot analyzes using the 5' end of the cDNA showed 11 mRNA species from 0.7 to 12 kb in size. However, the only two RNAs of 6 and 12kb size out of the multiple RNAs were hybridized to the most 3' end cDNA.The date clearly support the conclusion the cDNAs encode a collagen alpha chain which is different from known alpha chain. We propose to designate this collagen chain as alpha1(XIX) chain.

1)人α 1（XI）胶原基因启动子的分析我们克隆了人COL 11 A1基因的5'端部分，并确定了其主要转录起始位点的位置。直接上游序列的分析显示缺乏TATA基序和存在几个GC盒。与这一发现相一致，引物延伸和RNA酶保护测定确定了几个次要的转录起始位点周围的一个主要的，318 bp的上游从ATG密码子。瞬时转染实验表明，该启动子上游1.6kb区域具有启动子的能力。缺失构建体将其转录活性的序列缩小在核苷酸-541至-199之间。在该区域中，存在含有与AP-3结合位点相似的序列的单个足迹区域。2）新的人胶原蛋白链的一级结构和基因表达为了确定该链的完整一级结构，我们从人横纹肌肉瘤细胞系（CCL 136）的随机引物cDNA文库中分离了重叠克隆，推导的多肽含有一个1142个氨基酸残基的开放阅读框架。预测的多肽由COL 1至COL 5的五个胶原结构域组成。在结构上，这种胶原蛋白链最类似于α 1（XVI）链。利用cDNA的5'末端进行的北方印迹分析显示，大小为0.7 - 12 kb的11种mRNA。然而，在多个RNA中，只有两个大小为6和12 kb的RNA与最3'端的cDNA杂交。该数据清楚地支持cDNA编码不同于已知α链的胶原α链的结论。我们建议将这种胶原蛋白链命名为α 1（XIX）链。